This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To compare six novel vaccine regimens to induce CD4+ and CD8+ T Cells against SIV epitopes. Recent failures in clinical HIV vaccines have underscored the importance of more thoroughly evaluating basic science of HIV as well as testing new vaccine regimens and vectors. In an effort to overcome the limitations of more traditional vector-based vaccines, our laboratory has developed several novel immunization strategies. These new methods will allow us to directly prime specific T cell responses in a manner that previously has been impossible with other vaccine vectors and regimens. This should allow us to dissect the contributions of specific T cell responses in the control of SIV replication -- for the roles of both subdominant CD8+ T cells and virus-specific CD4+ T cells. In the R21 phase of this grant, we will compare six novel vaccination regimens: peptide-pulsed dendritic cells, peptide-conjugated nanobeads, peptide-pulsed PBMC, SIV peptides fused to a Hepatitis B core antigen (HBcAg) carrier gene, electroporated DNA+IL-12 and Adenovirus5. PROGRESS: We have completed vaccinating animals in groups one and two of the R21 phase. These animals received autologous dendritic cells pulsed with three Mamu-DRbw*606-restricted CD4 epitopes and primed with either autologous PBMC pulsed with the same epitopes or peptide-conjugated nanobeads. Two weeks after the final boosts, no animals made any detectable epitope-specific CD4+ T cell responses as detected by ELISPOT or ICS. The HBcAg vectors were completed by Dr. Deborah Fuller with each of the selected Mamu-A*01-restricted CD8 epitopes inserted into individual vectors. Six Mamu-A*01+ animals, groups three and four, received five doses of the HBcAg vector. After the fifth dose, no animals made any detectable epitope-specific CD8+ T cell response;however, they did respond well to Hepatitis peptide provided by Dr. Fuller indicating that they did receive doses of the vaccine. One month after the final prime, one group received autologous PBMC pulsed with each of the A*01 epitopes, while the other group received the A*01 epitopes conjugated to nanobeads. After boosting these animals, we still saw no detectable epitope-specific CD8+ T cell responses. Additionally, we made DNA and Adenovirus5 vectors containing the three CD4 epitopes and the five CD8 epitopes. We vaccinated animals with these constructs in the hope of eliciting epitope-specific CD4+ and CD8+ T cell responses. Two weeks after the Ad5 boost, we detected high frequency SIV-specific CD4+ T cell responses against all three epitopes in the vaccine. Two of the three animals made responses to all three epitopes, while the third animal made responses to two of the three epitopes. All three animals that received the CD8 epitopes made a high frequency response to one epitope in Env: Env TL9. One animal made an additional Pol LV10-specific CD8+ T cell response. While the epitope-specific CD4 vaccination was successful, none of the tested vaccine regimens elicited several high frequency epitope-specific CD8+ T cell responses. A publication is in preparation.